体外构建人巨核祖细胞/胃癌细胞融合体生成血小板的研究  

In vitro generating of platelets from human megakaryocyte progenitor/gastric cancer cells fusion vaccine

作　　者：雷慧芬[1] 范娅涵[1] 孟强[1] 尹智平[1] 陆华[1] 蒋天伦[1] 赵树铭[1] 

机构地区：[1]第三军医大学西南医院输血科,重庆400038

出　　处：《中国输血杂志》2011年第4期294-298,共5页Chinese Journal of Blood Transfusion

基　　金：重庆市自然科学基金(CSTC2009BB5144);第三军医大学临床科研基金(2010XLC23)

摘　　要：目的构建脐血巨核祖细胞/胃癌SGC7901细胞融合体产生功能性血小板。方法免疫磁珠分选人脐带血CD34+细胞,通过RPMI1640培养基体外静态培养,加入定向分化因子TPO、FL、IL-3、IL-6产生巨核祖细胞;复苏胃癌SGC7901细胞;采用聚乙二醇(PEG)化学融合法制备巨核祖细胞/胃癌SGC7901细胞融合体,HAT筛选系统分选融合细胞,继续培养;流式细胞仪检测培养孔上层液中类血小板颗粒表面CD41、CD62p表达,血小板聚集试验、粘附试验检测血小板功能,瑞氏染色观察颗粒形态。结果免疫磁珠分选的CD34+细胞培养7 d可增殖80倍,与胃癌SGC7901细胞株经PEG化学融合法融合成巨核祖细胞/胃癌细胞,融合率为(36.91±1.08)%;融合细胞培养10 d可增殖4倍,产生(6.1±1.21)×106/ml的类血小板颗粒;瑞氏染色后观察,其形态与正常血小板相似。类血小板颗粒表达血小板特异性标志CD41及血小板活化后标志CD62p,与正常血小板比较,CD62p在类血小板颗粒中表达略降低:正常血小板(74.11±1.71)%、类血小板颗粒(71.04±1.64)%;CD41表达明显降低:正常血小板(88.83±3.26)%、类血小板颗粒(72.51±2.79)%(t=6.59,P<0.05);聚集试验表明具有正常血小板相似的聚集功能;血小板粘附试验:正常血小板(40.43±1.63%)、类血小板颗粒(35.53±4.06%)。结论通过PEG细胞化学融合法构建的脐带血巨核祖细胞/胃癌SGC7901细胞融合体能产生功能性血小板。Objective To investigate whether the fusion vaccine from umbilical cord blood megakaryocyte progenitor and SGC7901 gastric cancer cells could be used to produce the functional platelets.Methods With the adding of RPMI1640 medium,human umbilical cord blood CD34＋ cells were sorted by immune magnetic beads and cultivated statically,then the directional differentiation factor TPO,FL,IL-3,IL-6 were joined respectively to produce megakaryocyte progenitor.With the being recovered of the SGC7901 gastric cancer cells,the megakaryocyte progenitor was fused with SGC7901 gastric cancer cells by polyethylene glycol（PEG）,and the fusion cells were screened out by HAT/HT medium,then was cultivated continually.The CD41 and CD62p expression of the platelet-like particles isolated from the culture were checked by the fluorescence-activated cell sorter（FACS）.The function of platelet was investigated by platelet aggregation test and platelet adhesion test,and the shape of particle was observed by Rick′s dyeing.Results The CD34＋ cells sorted by immune magnetic beads can proliferate as much as 80 times after 7-days cultivation.The fusion rate of the megakaryocyte progenitor and gastric cancer cells by polyethylene glycol was 36.91±1.08%.At the day 10 of fusing,it could be observed that the most cells were round,individual and well growing.The fusion cells can be proliferated as much as 4 times,and produced platelet-like particles of（6.1±1.21）×106/mL.It could also be observed similar the morphology as that of normal platelets by Rick′s dyeing.Compared the CD41 and CD62p expression of the platelet-like particles with normal platelets,it could be found that CD62p at the platelet-like particles decreased slightly（P0.05）,from（74.11±1.71）% to（71.04±1.64）%,there was no difference.But the CD41 expression was reduced significantly at the platelet-like particles（P0.05）,from（88.83±3.26）% of normal platelets to（72.51±2.79）% of platelet-like particles.The results of Rick′s dyeing showed that the

关 键 词：巨核祖细胞 脐带血 CD34+细胞 胃癌SGC7901细胞 融合体 血小板 聚乙二醇化学融合法 类血小板颗粒 

分 类 号：R446.1[医药卫生—诊断学] F331.143[医药卫生—临床医学]